Development of multivalent, ultrapotent nanobody cocktails for SARS-CoV-2 neutralization

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PROJECT SUMMARY/ABSTRACT The outbreak of COVID-19 has severely impacted global health and the economy. Cost-effective, highly efficacious therapeutics are urgently needed. Camelid VHH antibodies or nanobodies (Nbs) are small, highly stable, easily bioengineered, and can be rapidly and economically manufactured from microbes. They are highly robust and are flexible for administration, including possible delivery by nebulization. Together these unique properties of Nbs make their uses against respiratory viruses such as SARS-CoV-2 especially appealing. We recently developed a disruptive proteomic technology for large-scale identification of multi-epitope, drug- quality Nbs (Xiang et. al, Cell Systems. 2021). Using this technology, we identified > 8,000 high-affinity Nbs for the SARS-CoV-2 spike (S) receptor-binding domain (RBD) including Nbs that target highly neutralizing epitopes with sub-pM affinities and can neutralize SARS-CoV-2 at sub-ng/ml concentrations, which are unprecedented for antiviral antibody fragments. Structural proteomics revealed multiple distinct epitopes and potential neutralization mechanisms. Bioengineering of multi-epitope and multivalent constructs improved the potency to below 0.1 ng/ml (Xiang, et. al, Science. 2020). Most recently, we have demonstrated the high preclinical efficacy of an ultrapotent and stable trimeric Nb construct (PiN-21) for inhalation treatment of SARS- CoV-2 infection in a sensitive COVID-19 model (Nambulli, et. al, Science Advances. 2021). Intranasal delivery of PiN-21 at 0.6 mg/kg substantially reduces viral burdens in both airways. Critically, aerosol delivery of PiN-21 at 0.2 mg/kg decreases lung viral titers by 6-logs, minimizing lung pathology post-infection and preventing viral pneumonia. Combined with the marked stability and low production cost, this innovative therapy may provide a convenient and cost-effective option to mitigate the evolving pandemic and future events. In the revision, we aim to identify and characterize highly potent Nbs that are highly resistant to the variants of concern (VOCs) of SARS-CoV-2, investigate the neutralization mechanisms by structural approaches, and develop ultrapotent Nb constructs into safe and effective therapeutics. Our central hypothesis is that Nbs can be bioengineered into multivalent and ultrapotent forms to resist the mutational escape and the variants of concerns (VOCs) of SARS-CoV-2. Completion of our proposed studies will lead to cost-effective and convenient COVID-19 therapeutic candidates for translation into clinical trials. High-resolution structural studies will provide critical insights into how Nbs uniquely target the virus for high-affinity binding and neutralization. Critically, this project will serve as the testbed of our multidisciplinary platform to develop potent therapeutic and diagnostic reagents for future pandemics caused by coronaviruses or other pathogens.